Rational design of bifunctional protein degrader drugs

双功能蛋白降解药物的合理设计

• 批准号: 10378712
• 负责人: Karteek Kadimisetty
• 金额: $ 73.36万
• 依托单位: LIFESENSORS, INC.
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10378712
• 关键词: Autophagocytosis; Binding; Biochemical; Biological Assay; Biology; Biotechnology; C-terminal; Cell Line; Cell physiology; Cells; Chemicals; Clinic; Cullin Proteins; Development; Enzymes; Evaluation; Excision; Family; Genes; Glycine; Goals; Human Genome; In Vitro; Lead; Ligands; Ligase; Luciferases; Lysine; MDM2 gene; Mediating; Metabolism; Methodology; Methods; Monitor; Morphologic artifacts; Neurons; Nuclear Receptors; Pharmaceutical Preparations; Phase; Phosphotransferases; Play; Polyubiquitin; Process; Progress Reports; Promega; Property; Protac; Proteins; Proteomics; Reporter; Research; Ring Finger Domain; Role; Specificity; Structure-Activity Relationship; System; Testing; Therapeutic; Time; Tissues; Translating; Ubiquitin; Ubiquitination; Western Blotting; alpha synuclein; amino group; base; carboxylate; chimera drug; clinical development; commercialization; design; drug candidate; drug development; drug discovery; in vivo; live cell imaging; member; multicatalytic endopeptidase complex; mutant; novel drug class; novel therapeutic intervention; overexpression; protein degradation; protein function; protein transport; rapid test; rational design; receptor recycling; reconstitution; scale up; screening; small molecule; success; tau Proteins; transcription factor; ubiquitin ligase; ubiquitin-protein ligase

Ubiquitin (Ub) tags regulate multiple properties and functions of proteins in cells. Proteasomal degradation of target proteins is a well-established means whereby the Ub proteasome system (UPS) controls protein content. Enzymes called Ub E3 ligases conjugate Ub to target proteins by forming an isopeptide bond between the ε- amino group of the target protein lysine and the carboxylate of the C-terminal glycine of Ub. Conjugation of multiple Ubs forms poly-Ub chains at any of its seven lysines (K), and K6, K11, K27, K29, K33, K48, and K63 Ub chains having various roles are present in all tissues. Recently, several groups designed small molecules that bind to an E3 ligase at one end and a target protein at the other, physically facilitating ubiquitylation of the target protein, which is then degraded. This hijacking of a ligase to ubiquitylate a desired protein has launched a new class of drug called PROTACs (PROteolysis TArgeting Chimeras). PROTAC-based approaches for therapeutics offer several advantages: 1) selective, catalytic degradation of the target; 2) conversion of weak binders into selective PROTAC drugs; 3) degradation of overexpressed or mutant targets; and 4) maximal degradation from limited target engagement. To date, cereblon and VHL ligase binders have been most commonly used as vehicles to ubiquitylate target proteins such as nuclear receptors, kinases, transcription factors, and neuronal proteins tau and α-synuclein. A major problem has hindered development of new PROTAC drugs, however. Chemical optimization of PROTAC molecules depends on rapid evaluation of synthesized compounds to guide the synthetic strategy for producing drug candidate molecules. Assays currently available are labor intensive and do not provide results to the medicinal chemists fast enough – often, a week is required. In Phase I, a facile in vitro method employing Ub ligases cereblon and HDM2 was developed to screen for potential PROTAC drugs; PROTAC-mediated ubiquitylation of selected proteins was recapitulated in vitro in a way that mimics observed PROTAC-dependent ubiquitination and degradation of these proteins in vivo, achieving the aims of Phase I. In phase II, the utility of this method will be expanded to include representative members of all Ub ligase families (cullin families, RING finger ligases, Hect family ligases, and SUMO ligase), increasing the biochemical and chemical space for PROTAC drug discovery. To scale up PROTAC screens, a microtiter plate-based, high throughput method will be established to monitor in vitro PROTAC drug discovery, and biochemical and Ub mass spec proteomics will be employed to demonstrate that target protein lysines ubiquitylated in vitro are correlated with in vivo PROTAC mediated degradation of target proteins. Commercialization of the microtiter plate based PROTAC system will have a major impact on academic research as well as PROTAC drug discovery.

泛素 (Ub) 标签调节细胞中蛋白质的多种特性和功能。蛋白酶体降解 目标蛋白是 Ub 蛋白酶体系统 (UPS) 控制蛋白质含量的一种行之有效的方法。 称为 Ub E3 连接酶的酶通过在 ε- 之间形成异肽键，将 Ub 与靶蛋白结合。 目标蛋白赖氨酸的氨基和Ub C端甘氨酸的羧酸盐。共轭 多个 Ub 在其七个赖氨酸 (K)、K6、K11、K27、K29、K33、K48 和 K63 中的任何一个处形成多聚 Ub 链 具有各种作用的UB链存在于所有组织中。最近，几个团队设计了小分子 一端与 E3 连接酶结合，另一端与靶蛋白结合，以物理方式促进 E3 连接酶的泛素化 目标蛋白质，然后被降解。这种劫持连接酶来泛素化所需蛋白质的技术已经启动 一类新的药物称为 PROTAC（蛋白水解靶向嵌合体）。基于 PROTAC 的方法 疗法具有以下几个优点：1）选择性、催化降解靶标； 2）弱转换 选择性 PROTAC 药物的粘合剂； 3) 过度表达或突变靶标的降解； 4) 最大 有限的目标参与导致的退化。迄今为止，cereblon 和 VHL 连接酶结合剂是最 通常用作泛素化靶蛋白（如核受体、激酶、转录）的载体 因子、神经元蛋白 tau 和 α-突触核蛋白。一个重大问题阻碍了新 PROTAC 的开发 然而，药物。 PROTAC 分子的化学优化取决于合成的快速评估 指导生产候选药物分子的合成策略的化合物。目前可用的检测方法 是劳动密集型的，并且不能足够快地向药物化学家提供结果——通常需要一周的时间。 在第一阶段，开发了一种使用 Ub 连接酶 cereblon 和 HDM2 的简便体外方法来筛选 潜在的PROTAC药物； PROTAC 介导的选定蛋白质的泛素化在体外重现 模拟体内观察到的 PROTAC 依赖性泛素化和这些蛋白质降解的方式， 实现第一阶段的目标。在第二阶段，该方法的效用将扩大到包括代表性 所有 Ub 连接酶家族（cullin 家族、RING Finger 连接酶、Hect 家族连接酶和 SUMO 连接酶）的成员， 增加 PROTAC 药物发现的生化和化学空间。为了扩大 PROTAC 屏幕的尺寸， 将建立基于微量滴定板的高通量方法来监测体外 PROTAC 药物发现， 将采用生化和 Ub 质谱蛋白质组学来证明目标蛋白赖氨酸 体外泛素化与体内 PROTAC 介导的靶蛋白降解相关。 基于微量滴定板的 PROTAC 系统的商业化将对学术研究产生重大影响 以及 PROTAC 药物发现。